Through its regulation of LH and FSH release, gonadotropin releasing hormone (GnRH/LHRH) establishes and modulates the neuroendocrine aspects of the reproductive cycle. Because those factors governing the expression of this hormone are critical to the subsequent coordination of these activities, the objective of this proposal is to define those processes involved in the biosynthesis of GnRH. The DNA sequence coding for GnRH will be isolated using recombinant DNA technology and used to study the synthesis of GnRH mRNA in hypothalamic tissue. Specifically, a synthetic oligonucleotide whose sequence is predicted by the amino acid sequence of the protein will be used to isolate a cloned DNA sequence encoding the precursor of GnRH. Serving as an analytical probe for the presence of GnRH-specific mRNA, this cDNA sequence will permit quantitation of the minute amounts of hypothalamic GnRH synthesis in a highly sensitive filter hybridization assay. Additionally in situ hybridization with the GnRH cDNA probe will, by identification of the GnRH mRNA, distinguish those cells synthesizing GnRH de novo. The production of GnRH mRNA in mid and anterior hypothalamic regions will be investigated to determine whether the tonic and pulsatile release modes reflect differential biosynthesis of the hormone during the rat estrus cycle, i.e. whether the cyclical production of GnRH is regulated at the level of the expression of its gene. With this information as a framework, the GnRH cDNA probe will be used to examine the influences of the gonadal steriod hormones estrogen and progesterone on the synthesis of GnRH mRNA. Because GnRH has been so extensively characterized physiologically, anatomically, and biochemically, the techniques of molecular biology may now be applied to some fundamental but as yet unresolved questions regarding GnRH biosynthesis. In addition to confirming its synthesis in a precursor form and localizing the exact sites of its synthesis, those aspects of GnRH production that are regulated at the level of gene expression can now be ascertained. By initially characterizing the production of GnRH in normal rats, it will be possible through a similar approach to assess the regulatory role of GnRH in reproductive events, e.g. during fetal development, puberty and pregnancy.